RESUMEN
Glutamate is a major neurotransmitter in the central nervous system, and abnormal glutamate neurotransmission has been implicated in many neurological disorders, including schizophrenia, Alzheimer's disease, Parkinson's disease, addiction, anxiety, depression, epilepsy, and pain. Metabotropic glutamate receptors (mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner. In the present study, we further characterize the human mGluR2 (hmGluR2) potentiator binding site by showing that the substitution of the three amino acids found to be required for hmGluR2 potentiation, specifically Ser(688), Gly(689), and Asn(735), with the homologous hmGluR3 amino acids, inactivates the positive allosteric modulator activity of several structurally unique mGluR2 potentiators. Based on the characterization of the hmGluR2 potentiator binding site, we developed a novel scintillation proximity assay that was able to discriminate between compounds that were hmGluR2-specific potentiators, and those that were active on both hmGluR2 and hmGluR3. In addition, we substituted Ser(688), Gly(689), and Asn(735) into hmGluR3 and created an active hmGluR2 allosteric modulation site on the hmGluR3 receptor.
Asunto(s)
Sitio Alostérico/genética , Aminoácidos/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Regulación Alostérica/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Animales , Línea Celular , Células Cultivadas , Humanos , Masculino , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica/genética , Ratas , Ratas Sprague-DawleyRESUMEN
The receptor localization of metabotropic glutamate receptors (mGlu) 2 and 3 was examined by using in situ hybridization and a well-characterized mGlu2-selective antibody in the rat forebrain. mGlu2 was highly and discretely expressed in cell bodies in almost all of the key regions of the limbic system in the forebrain, including the midline and intralaminar structures of the thalamus, the association cortices, the dentate gyrus of the hippocampus, the medial mammillary nucleus, and the lateral and basolateral nuclei of the amygdala. Moreover, presynaptic mGlu2 terminals were found in most of the forebrain structures, especially in the lateral part of the central nucleus of the amygdala, and the CA1 region of the hippocampus. Although some overlaps exist, such as in the hippocampus and the amygdala, the expression of mGlu3 mRNA, however, appeared to be more disperse, compared with that of mGlu2 mRNA. These distribution results support previous behavioral studies that the mGlu2 and 3 receptors may play important roles in emotional responses. In addition to its expression in glia, mGlu3 was distinctively expressed in cells in the GABAergic reticular nucleus of the thalamus. Local infusion of a non-selective mGlu2/3 agonist, LY379268, in the reticular nucleus of the thalamus, significantly reduced GABA release, suggesting that mGlu3 may also play a role in central disinhibition.
Asunto(s)
Prosencéfalo/metabolismo , Receptores de Glutamato Metabotrópico/biosíntesis , Animales , Western Blotting , Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Microdiálisis , Microscopía Confocal , ARN Mensajero/análisis , Ratas , TransfecciónRESUMEN
Metabotropic glutamate receptor 2 (mGluR2) has been implicated in a variety of CNS disorders, including schizophrenia. Disclosed herein is the development of a new series of allosteric potentiators of mGluR2. Structure-activity relationship studies in conjunction with pharmacokinetic data led to the discovery of indole 5, which is active in an animal model for schizophrenia.
Asunto(s)
Acetofenonas/farmacología , Modelos Animales de Enfermedad , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismo , Acetofenonas/química , Acetofenonas/farmacocinética , Regulación Alostérica/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Reactivos de Enlaces Cruzados/química , Humanos , Ketamina/farmacología , Estructura Molecular , Ratas , Esquizofrenia/inducido químicamente , Relación Estructura-ActividadRESUMEN
We have identified and synthesized a series of biphenyl-carboxylic acid indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency and the brain to plasma ratio of the initial lead led to the discovery of 5 and 23 (EC50=111 and 5 nM, respectively).
Asunto(s)
Compuestos de Bifenilo/síntesis química , Indanos/síntesis química , Receptores de Glutamato Metabotrópico/agonistas , Regulación Alostérica , Animales , Compuestos de Bifenilo/metabolismo , Compuestos de Bifenilo/farmacocinética , Química Encefálica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Indanos/metabolismo , Indanos/farmacocinética , Ratas , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamiento farmacológico , Relación Estructura-Actividad , Distribución TisularRESUMEN
We have identified and synthesized a series of phenyl-tetrazolyl and 4-thiopyridyl indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation, as well as PK properties, led to the discovery of 28 (EC50=186 nM), which displayed activity in a rodent model for schizophrenia.
Asunto(s)
Indanos/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Regulación Alostérica , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Indanos/farmacocinética , Modelos Químicos , Estructura Molecular , Unión Proteica , Ratas , Esquizofrenia/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
We have identified and synthesized a series of 4-thiopyridyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward replacement of the tetrazole in the initial lead led to the discovery of 16 (EC(50)=340 nM), which showed improved brain penetration over the initial lead.
Asunto(s)
Acetofenonas/metabolismo , Piridinas/química , Piridinas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Acetofenonas/química , Regulación Alostérica/fisiología , Animales , Encéfalo/metabolismo , Línea Celular , Humanos , Unión Proteica/fisiología , Ratas , Receptores de Glutamato Metabotrópico/agonistas , Relación Estructura-Actividad , TetrazolesRESUMEN
We have identified and synthesized a series of aryl-tetrazoyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation led to the discovery of 22 (EC(50)=93nM, 128% potentiation).
Asunto(s)
Acetofenonas/síntesis química , Receptores de Glutamato Metabotrópico/agonistas , Tetrazoles/síntesis química , Acetofenonas/química , Acetofenonas/farmacología , Regulación Alostérica , Estereoisomerismo , Relación Estructura-Actividad , Tetrazoles/química , Tetrazoles/farmacologíaRESUMEN
Herein we disclose the discovery of a new class of positive allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2), phenyl-tetrazolyl acetophenones, e.g. 1-(2-hydroxy-3-propyl-4-[4-[4-(2H-tetrazol-5-yl)phenoxy]butoxy]phenyl) ethanone (4). These potentiators were shown to have no effect in the absence of glutamate as well as no effect at mGlu3 or the other mGlu receptors. The compounds were also evaluated in rodent models with potential relevance for schizophrenia, and 4 was shown to have activity in the inhibition of ketamine-induced norepinephrine release and ketamine-induced hyperactivity. This represents the first example of the efficacy of mGlu2 receptor potentiators in these models.
Asunto(s)
Acetofenonas/síntesis química , Acetofenonas/farmacología , Regulación Alostérica , Receptores de Glutamato Metabotrópico/agonistas , Animales , Conducta/efectos de los fármacos , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Glutámico/farmacología , Hipercinesia/tratamiento farmacológico , Norepinefrina/metabolismo , Ratas , Esquizofrenia/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
In the present study, we describe the characterization of a positive allosteric modulator at metabotropic glutamate subtype 2 receptors (mGluR2). N-(4-(2-Methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379) is a selective positive allosteric modulator at human mGluR2 and is without activity at human mGluR3. Furthermore, LY487379 has no intrinsic agonist or antagonist activity at hmGluR2, as determined by functional guanosine 5'(gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding, single-cell Ca2+ imaging, and electrophysiological studies. However, LY487379 markedly potentiated glutamate-stimulated [35S]GTPgammaS binding in a concentration-dependent manner at hmGluR2, shifting the glutamate dose-response curve leftward by 3-fold and increasing the maximum levels of [35S]GTPgammaS stimulation. This effect of LY487479 was also observed to a greater extent on the concentration-response curves to selective hmGluR2/3 agonists. In radioligand binding studies to rat cortical membranes, LY487379 increased the affinity of the radiolabeled agonist, [3H]DCG-IV, without affecting the binding affinity of the radiolabeled antagonist, [3H]LY341495. In rat hippocampal slices, coapplication of LY487379 potentiated synaptically evoked mGluR2 responses. Finally, to elucidate the site of action, we systematically exchanged segments and single amino acids between hmGluR2 and hmGluR3. Substitution of Ser688 and/or Gly689 in transmembrane IV along with Asn735 located in transmembrane segment V, with the homologous amino acids of hmGluR3, completely eliminated LY487379 allosteric modulation of hmGluR2. We propose that this allosteric binding site defines a pocket that is different from the orthosteric site located in the amino terminal domain.